Biomedical subjects
Publications and source records attributed to H Degani.
31P NMR spectroscopy was utilized to study phosphate metabolism of perfused ovaries. In luteinized ovaries, a loss in ATP of approximately 30% with a concomitant acidification was reversed by 50 microM adenosine. The results suggest that in the ovaries, adenosine can serve to regulate ATP concentration during periodic hypoxic challenges.
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Differences in the energetics of breast carcinomas and benign breast tumors were monitored by recording their high-energy phosphate concentrations using phosphorus-31 magnetic resonance (MR) spectroscopy. Analysis of the P-31 spectra of biopsy samples from five benign breast tumors and nine breast carcinomas showed that the concentrations of nucleoside triphosphates and phosphomonoesters are consistently higher in the carcinomas than in the benign tumors by an average factor of about three. In addition, the chemical shift differences between the alpha-adenosine triphosphate (alpha-ATP) and beta-ATP signals and between the gamma-ATP and beta-ATP signals were larger by about 0.2 ppm in the carcinomas. These differences result from the lower fraction of magnesium-bound ATP found in the carcinomas under our experimental conditions and reflect a decrease of about two in the concentration of free magnesium ions in the carcinomas relative to that in the benign tumors. Despite the limited number of tumors studied, the results suggest that in vivo P-31 MR spectroscopy may become a reliable method for the diagnosis of breast tumors.
The kinetics of the phosphate exchange by creatine kinase (CK) was studied in solution and in the Langendorff-perfused rat heart at 37 degrees C. 31P inversion-transfer (IT) and saturation-transfer (ST) methods were applied. The kinetic parameters obtained by the two magnetization transfer methods were the same, whether in solution or in the perfused heart. Inversion transfer is the more efficient method, yielding the kinetic constants for the exchange and the relaxation rates of the transferred phosphate in both substrates, in one experiment. In solution the forward (kF) and reverse (kR) pseudo-first-order rate constants for the CK reaction (kF = k1[MgADP][H+]; kR = k-1[creatine]) as well as the concentrations of phosphocreatine (PCr), MgATP, and creatine (Cr) remained constant between pH 6.9 and pH 7.8. Equilibrium at this pH region is therefore maintained by compensating changes in the concentration of MgADP. The forward and reverse fluxes in the perfused heart were equal with an average flux ratio (fluxF/fluxR) of 0.975 +/- 0.065 obtained by both methods. Average values of kF and kR were 0.725 +/- 0.077 and 1.12 +/- 0.14 s-1, respectively. These results clearly indicate that the CK reaction in the Langendorff-perfused heart is in equilibrium and its rate is not limited by the diffusion of substrates between different locations of the enzyme. There is therefore no indication of compartmentation of substrates of the CK reaction.
The exchange rate and enthalpy and entropy of activation of the diffusion of the first five n-alkylamines across egg phosphatidylcholine vesicles has been measured by 1H-NMR spectroscopy employing the 1:2 Gd3+-EDTA complex as a relaxation reagent. The permeability determined from the exchange rate of the ethyl through the pentyl derivatives increased sequentially with increasing chain length from 7.10(-7) to 4.10(-4) cm/s, respectively, at 25 degrees C. The permeability of methylamine was similar to that of ethylamine (1.10(-6) cm/s at 25 degrees C) and exhibited a relatively smaller entropy increase. The enthalpy of activation for the transfer reaction was high for all amine derivatives (20 kcal/mol). The entropy of activation increased with increasing chain length. The results indicate that the rate of diffusion is dominated by the partition into the membrane. Methylamine, being the smallest molecule in this series, can probably diffuse also through vacancies formed by the internal motions of the lipid chains.
The metabolic states and the uptake and metabolism of [1-(13)C]glucose, [2-(13)C]glycine, and [(15)N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by (13)C and (15)N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH(4) (+) assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.
Changes in the concentrations of high-energy phosphate metabolites were measured by 31P NMR spectroscopy of surviving rat uteri from 0-48 h following estrogen administration. Concentrations (millimoles per kilogram wet weight) of these metabolites in the untreated immature uterus, measured at 4 degrees C, were found to be the following: creatine phosphate (CP), 2.1 +/- 0.2; nucleoside triphosphates, mainly adenosine 5'-triphosphate (ATP), 4.6 +/- 0.4; phospho monoesters, primarily sugar phosphates (SP), 5.4 +/- 0.7; and inorganic phosphate (Pi), 0.8 +/- 0.4. Adenosine 5'-diphosphate (ADP) concentration was estimated to be approximately 40 mumol/kg wet weight from the assumed equilibrium of the creatine kinase reaction. The concentration of CP, and to lesser extent ATP and SP, declined within the first 1.5-3 h after injection of 17 beta-estradiol, returned to control values between 6 and 12 h, and then increased, reaching maximal concentrations at 24 h. From the fractions of the total soluble ATP in free and Mg2+-bound forms, [free Mg2+] in the untreated uterus was estimated to be 0.2-0.4 mmol/kg wet weight. An increase in [free Mg2+] in the uterus was detected 1.5 h after estrogen injection. A subsequent parallel increase in the ratio of ATP to CP concentrations suggests that estrogen can also affect the apparent creatine kinase equilibrium by modulating [free Mg2+].
Using 31P-NMR spectroscopy, we have investigated possible involvement of metabolic processes in the lysis of nucleated cells induced by low levels of antibody to cell surface antigens and complement. Within 10 min of antibody plus complement attack, before onset of overt lysis, we have observed a marked, selective reduction in the intracellular content of phosphocreatine and adenosine triphosphate (ATP). A longer attack is accompanied by total depletion of either phosphocreatine or ATP in residual cells which preserved other phosphate compounds. The results indicate that in nucleated cells formation of putative complement-dependent membrane channels induces exhaustive hydrolysis of ATP. It is suggested that ATP deprivation could in turn lead to colloid-osmotic swelling, membrane rupture, and cell death.
The shift reagent dysprosium tripolyphosphate [Dy(PPPi)7-2] has been applied to the nuclear magnetic resonance (NMR) study of 23Na in frog skin. The anion complex produces slight increases in the transcellular short-circuit current and conductance of the tissue. However, the electrophysiological responsiveness of the tissue to vasopressin, amiloride, and transient removal of extracellular potassium appear unimpaired. A large signal of [31P]phosphocreatine was also observed, further documenting that relatively little damage was produced by the reagent. Three signals of 23Na were observed, reflecting the sodium pools in an external standard solution, the extracellular space, and a medium relatively inaccessible to Dy(PPPi)7-2. The size of the reagent-insensitive signal could be increased by ouabain, a selective inhibitor of sodium extrusion, and could be decreased by amiloride, a selective inhibitor of sodium entry. The results indicate that the anion complex-insensitive signal reflects, at least in part, intracellular sodium and suggest that 23Na may become a useful complementary tool for monitoring the intracellular sodium content of transporting epithelia.
Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T1 relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (delta H not equal to ) were determined for glycerol at 25 degree C: for phosphatidylcholine vesicles 5 . 10-6 cm/s and 11 +/- 2 kcal/mol; for pig erythrocytes 7 . 10-8 cm/s and 18 +/- 3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17 degrees C was found to be exceptionally low and only a lower limit (P less than 5 . 10-11 cm/s) could be calculated. At temperatures above 50 degree C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.
T1 nuclear relaxation measurements of 1H and 17O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25 degree C were found to be 1.5.10-3 cm/s and 1.8.10-3 cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165-173.
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We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the 1H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, S(t), can be approximated by the simple first-order rate law: S(t) = S(0) exp(-k't), where k' is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of k' on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of k' on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of k' on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.
Nuclear relaxation measurements of 1H and 17O of water have been applied to study the kinetics of water diffusion across vesicular lipid membranes. Differentiation between the intra- and extravesicular media was achieved by entrapping Mn2+ inside the vesicles. The water permeability of egg phosphatidylcholine vesicles was found to be 2.9 x 10(-3) cm/s at 25 degrees C, with an activation energy of 10.5 kcal/mol which remains constant through the temperature range 0-65 degrees C. The water permeability across vesicular bilayers of L-alpha-dipalmitoyl phosphatidylcholine exhibited a sharp change through the lipid phase transition. The permeability in the lipid crystalline phase (45 degrees C) was found to be 7.2 x 10(-3) cm/s with an activation energy of 7.2 kcal/mol. Below the transition at the gel phase (35 degrees C) a permeability of 1.0 x 10(-3) cm/s was determined. The results indicate that water diffuses through lipid membranes in the liquid crystalline phase in a similar fashion to its diffusion in hydrocarbon liquids. However, when the lipids undergo a phase transition to the gel state, this similarity does not hold any more and water diffusion becomes much more restricted than in hydrocarbon liquids. The change in water permeability through the phase transition was correlated with the changes observed in the lipid segmental motion determined from 13C T1 measurements.
The linear peptide hormones angiotensin II and [Asn1, Val5]angiotensin II are found to mediate the transport of Mn(II) ions across phosphatidylcholine bilayers. Nuclear magnetic resonance spectroscopy (NMR) is applied to monitor the rate of transport of Mn(II) ions by measuring the rate of disappearance of the 1H NMR signal of the choline methyl groups of the inner phospholipid layer. This rate of disappearance is analyzed in terms of a pseudo-first-order rate equation for the transport process. The rate of transport of Mn(II) varies linearly with both the concentrations of Mn(II) and agiotensin II (A-II) present, suggesting that the ions are transported in a complex with 1:1 stoichiometry. An analysis of the temperature dependence of the rate of transport yielded an energy of activation of 29 +/- 5 kcal/mol and an entrophy of activation of 10 eu for the transport process. The activation parameters are discussed in terms of defining the rate-limiting step in the transport process. The pH dependence of the hormone-mediated rate of Mn(II) transport is similar to the pH dependence of the metal complexation process measured in a separate study. The presence of La(III) or Tris decelerates the rate of Mn(II) transport by presumably competing with either Mn(II) or A-II, respectively, in the binding process. On the basis of the results presented here as well as literature data, we suggest that the ionophoric properties of these two hormones may be relevant to understanding the role of metal ions in their physiological activities.
The thermotropic behavior and the dynamic properties of the polar lipids of Halobacterium halobium were studied by 1H and 13C NMR. The studies were performed on three different preparations: micellar solutions in deuterated chloroform, multilamellar dispersions in 2H2O, and unilamellar vesicles obtained from the latter by ultrasonication. Due to the methyl side groups in the alkyl chains of these lipids, the 13C spectra are highly resolved in particular in the micellar solution, allowing peak assignment of the alkyl chain carbons. Proton and 13C T1 relaxation and line width measurements were made for all three preparations. In both lamellar dispersions, sharp discontinuities in these parameters were observed around 35 degrees C. This behavior is attributed to an abrupt change in the dynamics of the lipids within the membrane. The variation of the specific relaxation rate 1/NT1 of the 13C nuclei along the hydrocarbon chains of the H. halobium lipids exhibits maxima at the tertiary carbons to which the methyl side groups are attached. These maxima are particularly pronounced in the lamellar phases and suggest that the segmental motion ("kink" formation) at the tertiary carbons is hindered by the presence of the methyl groups.
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Nuclear magnetic resonance spectroscopy has been applied as a method for studying manganous ions transport across the membrane of phosphatidylcholine vesicles. The rates of the ionophore X-537A (lasalocid A)-mediated Mn2+ transport have been measured as a function of ionophore concentration, pH of the vesicle suspension, and temperature. The translocation was found to occur via a neutral complex composed of one manganous ion bound in two ionized X-537A molecules (Mn X2). The activation energy for the overall transport process was determined to be 22 +/- 5 kcal/mol. Also a pKa of 5.0 +/- 0.2 was determined for the ionophore acid dissociation equilibrium in the vesicle suspension.